Clonal hematopoiesis driven by DNMT3A mutations fuels a vicious cycle of cardiac inflammation and fibrosis in heart failure
Authors: M Shumliakivska, W Abplanalp, G Luxan, B Schuhmacher, R Schulze Bruening, D John, S Cremer, A Zeiher, S Dimmeler
Journal: Cardiovascular Research (2022)

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Age-acquired, non-malignant somatic mutations in epigenetic regulators lead to clonal hematopoiesis of indeterminate potential (CHIP). Mutations in the methylation regulator DNA methyltransferase 3 alpha (DNMT3A) were shown to be correlated with poor prognosis in patients with heart failure (HF). Although recent reports suggest increased inflammation in DNMT3A CHIP carriers, the paracrine interactions between CHIP immune cells and cardiac fibroblasts remain to be fully elucidated. Purpose To investigate mechanisms by which DNMT3A CHIP mutant macrophages facilitate the activation of fibroblasts and thus, the progression of pathological fibrosis in heart failure. Methods Using DB CellChat and CellPhone softwares on transcriptomic data from single cell and nuclei RNA sequencing, we performed interaction analysis of circulating monocytes obtained from HF patients with and without DNMT3A CHIP mutations and fibroblasts obtained from myocardial tissue specimens. In vitro validation of potential interactions was performed using siRNA-based loss of function studies and included macrophages-to-fibroblasts and fibroblasts-to-macrophages co-culture experiments. They were followed by immunocytochemistry analysis, gene and protein expression measurements. Results We demonstrated that the secretome from cardiac fibroblasts promoted the inflammatory profile by inducing M1 skewing, while reducing expression of anti-inflammatory M2 markers in DNMT3A-silenced macrophages in vitro (p&amp;lt;0.05). In addition, co-culture of fibroblasts with DNMT3A-silenced macrophages induced an increase in fibroblast activation, as evidenced by induction of smooth muscle actin expression (1.61-fold, p&amp;lt;0.05) and augmented collagen gel contraction (1.40-fold induction, p&amp;lt;0.05). Curiously, we identified an upregulated activin A receptor type I (ACVR1B)/activin A signalling pathway as a monocytes/macrophages-to-cardiac fibroblasts specific interaction for DNMT3A CHIP carriers. Indeed, DNMT3A variant allele frequency was positively correlated with ACVR1B gene expression levels (p&amp;lt;0.05). Moreover, we confirmed that ACVR1B is upregulated in monocytes of HF patients with loss-of-function DNMT3A CHIP mutations versus HF patients without these mutations (p&amp;lt;0.05). Importantly, silencing of DNMT3A in macrophages in vitro led to an increase in ACVR1B gene expression (p&amp;lt;0.05). Conclusions DNMT3A CHIP mutations in circulating monocytes augment macrophages-to-cardiac fibroblasts interactions in patients with HF. While DNMT3A CHIP monocytes/macrophages are sensitized to cardiac fibroblasts secretome and become more pro-inflammatory, conversely, the secretome of DNMT3A mutant cells leads to myofibroblastic activation of fibroblasts. These data support the hypothesis of a vicious cycle fueling cardiac fibrosis in HF progression of DNMT3A mutation carriers.